Chicken anemia virus vaccine from cell line

ABSTRACT

Provided is a chicken infectious anemia virus (CIAV) vaccine, comprising live CIAV passaged in MDCC-MSB-1 (MSB-1) cells, wherein the vaccine does not cause Marek&#39;s Disease. Also provided is a CIAV vaccine comprising a CIA virus having the sequence of SEQ ID NO: 1. A method of making a CIAV vaccine is provided, comprising culturing CIAV in MSB-1 cells, and removing or killing any Marek&#39;s disease virus present in the CIAV-containing MSB-1 culture. Provided a method of immunizing a chicken against CIAV infection, comprising administering to the chicken an amount of the CIAV vaccine of the invention sufficient to induce an immune response to CIAV.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority from U.S. ProvisionalApplication Ser. No. 60/317,239, filed Sep. 5, 2001, which applicationis hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The invention relates generally to a vaccine for chicken infectiousanemia virus, methods of making the vaccine and methods of immunizationusing the vaccine.

BACKGROUND

CIAV causes clinical and subclinical disease in chickens, and isrecognized as an important avian pathogen worldwide. In young chickens,CIAV causes a transient severe anemia due to destruction oferythroblastoid cells in the bone marrow and immunodeficiency due todepletion of cortical thymocytes. The depletion of cortical thymocytesis considered to cause a transient immunodeficiency resulting inenhanced concurrent infections and to vaccination failures. Thedepletion of thymocytes and most likely also of erythroblastoid cellsoccurs via VIAC-induced apoptosis.

CIAV is a small virus of a unique type with a particle diameter of 23-25nm and a genome consisting of a circular single-stranded (minus strand)DNA. This DNA multiplies in infected cells via a circulardouble-stranded replicative intermediate. CIAV is not related to otherknown animal single stranded circular DNA viruses, such as porcinecircovirus and psittacine beak-and-feather disease virus.

The major transcript from the CIAV genome is an unspliced polycistronicmRNA of about 2100 nucleotides encoding three proteins of 51.6 kDa(VP1), 24.0 kDa (VP2) and 13.6 kDa (VP3 or apoptin). All three proteinsare synthesized in CAIV-infected cells.

To reduce the economic damage caused by CIAV infection, it is necessaryto provide a cost-effective vaccine against CIAV. Prior attempts toprovide a CIAV vaccine have required the passaging and propagation ofCIAV in CIAV-susceptible SPF-embryos (See Vielitz and Voss,International Symposium on Infectious Bursal Disease and ChickenInfectious Anemia, Rauischholzhausen, Germany, 21-24 June 19114).Attempts to produce CIAV in cell lines has been problematic due toinfection of susceptible cell lines with Marek's disease virus. Thus, aneed exists for a vaccine produced in cultured cells that will not causeMarek's disease.

The present invention meets the needs of this field by providing avaccine without the disadvantages of embryo passaging and without thedisadvantages of Marek's disease virus contamination.

SUMMARY OF THE INVENTION

In accordance with the purpose(s) of this invention, as embodied andbroadly described herein, this invention, in one aspect, relates to achicken infectious anemia virus (CIAV) vaccine, comprising live CIAVpassaged in MDCC-MSB-1 (MSB-1) cells, wherein the vaccine does not causeMarek's Disease.

In another aspect, the invention provides a CIAV vaccine comprising aCIA virus having the sequence of SEQ ID NO: 1.

In another aspect, the invention provides a method of making a CIAVvaccine, comprising culturing CIAV in MSB-1 cells, and removing orkilling any Marek's disease virus present in the CIAV-containing MSB-1culture. The method can include subjecting the CIAV-containing MSB-1cell culture to at least 3 cycles of freezing and thawing, followed by astep of maintaining the cells for about 3 days at about. Alternatively,filtration may be used, or centrifugation followed by treatment at about37° C.

In a further aspect, the invention provides a method of immunizing achicken against CIAV infection, comprising administering to the chickenan amount of the CIAV vaccine of the invention sufficient to induce animmune response to CIAV.

The invention has the advantage that it provides a CIAV vaccine that canbe produced in a cell line and is free of contaminating viruses.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate (one) several embodiment(s) ofthe invention and together with the description, serve to explain theprinciples of the invention.

FIG. 1 shows PCR products (1=marker, 2=Del Ros, 3=Intervet CIAV embryoadapted and attenuated vaccine, 4=1:2 cells, 5=1:2 supernatant, 6=1:10cells, 7=1:10 supernatant, 8=MSB-1 cells only).

FIG. 2 shows restriction enzyme analysis with HindIII (1=marker, 2=CIAVDel Ros uncut, 3=CIAV Del Ros HindIII, 4=Intervet CIAV uncut, 5=IntervetCIAV HindIII, 6=1:2 Intervet CIAV uncut, 7=1:2 Intervet CIAV sampleHindIII, 8=1:10 Intervet CIAV HindIII).

FIG. 3 shows the effect of freeze-thaw on the viability of MDV (Rispen'svirus).

FIG. 4 shows the effect of 37° C. on the viability of MDV (Rispen'svirus) after 3 freeze-thaw cycles.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of preferred embodiments of the inventionand the Examples included therein and to the Figures and their previousand following description.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about” or“approximately,” it will be understood that the particular value formsanother embodiment. It will be further understood that the endpoints ofeach of the ranges are significant both in relation to the otherendpoint, and independently of the other endpoint.

The invention provides a chicken infectious anemia virus (CIAV) vaccine,comprising live CIAV passaged in MDCC-MSB-1 (MSB-1) cells, wherein thevaccine does not cause Marek's Disease.

The CIAV vaccine of the invention does not produce gross lesions in asignificant number of chicken embryos. The vaccine has been tested inembryos, and in the studies done, produces lesions in fewer than 10% ofembryos. This is in contrast to a different CIAV vaccine that isproduced in chicken embryos, and causes significant lesions in theembryos.

The CIAV vaccine of the invention also does not produce significantanemia in chicken embryos.

The invention provides a CIAV vaccine comprising of any of the reportedstrains (e.g., intervet strain, Cux-1 strain, Texas strain, DRP5 (DelRos after 5 passages), CAV-15 strain, etc.). For example, inventionprovides a CIAV vaccine comprising a CIAV having the sequence of SEQ IDNO: 1. This is the sequence the Del Ros strain. The invention alsoprovides a CIAV vaccine comprising any CIAV strain that is newlyisolated or is a modified form of a known strain.

A method of making a CIAV vaccine is provided, comprising culturing CIAVin MSB-1. In addition to providing a method of making MSB-1-culturedCIAV free of Marek's disease virus (MDV) (see below and Example 1), themethod can also produce CIAV to a titer of at least 10^(8.1). This is ahigher titer than is typically obtained for this virus in MSB-1 cells.The details of one example of this process are provided in Example 1. Itis recognized that other methods for culturing CIAV in MSB-1 cells maybe routinely developed and practiced.

The method of making a CIAV vaccine can be used with any of the reportedCIAV strains (e.g., intervet strain, Cux-1. strain, Texas strain, DRP5(Del Ros after 5 passages), CAV-15 strain, etc.). For example, themethod of making a CIAV vaccine can use a CIAV having the sequence ofSEQ ID NO: 1. The method of making a CIAV vaccine can also use any CIAVstrain that is newly isolated or is a modified form of a known strain.

The method of making a CIAV vaccine can further comprise the step ofseparating the cultured CIAV from the MSB-1 cells, which typicallycontain MDV. CIAV is secreted into the culture medium, thus allowing fora variety steps for separating the CIAV from MSB-1 cells. For example,the method of making a CIAV vaccine can comprise a step of subjectingthe CIAV to at least 3 cycles of freezing and thawing. This disrupts thecells and inactivates a substantial amount of the MDV (an obligateintracellular pathogen). This step is usually followed with a step ofmaintaining the cells for about 3 days at about 37° C. This inactivatesany remaining MDV. A further method of making the CIAV grown in MSB-1cells free of MDV can comprise the step of filtering thevirus-containing MSB-1 cells through a 5 micron filter. Filtering canrupture the cells because they fragile, and it also removes any intactcells. Examples of these processes for removing MDV from the CIAVvaccine and for killing any MDV in the CIAV culture are provided inExample 1 and Example 9). It is recognized that other methods forobtaining the CIAV vaccine from MSB-1 cells that is free of MDV may beroutinely developed and practiced. For example, a process ofcentrifuging the CIAV infected MSB-1 cells to remove cells and most ofthe MDV, followed by cycles of freeze-thaw of the supernatant andmaintenance at 37° C. to kill any remaining MDV is also effective. Thusthe methods of making the CIAV vaccine provided herein produce a vaccinethat does not cause Marek's disease in chickens immunized with thevaccine.

The invention provide a method of immunizing a chicken against CIAVinfection, comprising administering to the chicken an amount of the CIAVvaccine of the invention sufficient to induce an immune response toCIAV. The immune response produced is protective against infection byCIAV. Thus, the immune response is also protective against clinicaldisease caused by CIAV infection. Although the present CIAV vaccine isnot attenuated immunized chickens (e.g., hens) do not typically getsick, because of the recognized age-resistance to this virus.

The immunization method of the invention extends to the progeny of animmunized hen. The immune response in the hen produces antibodies in thehen that are passed to the chick through the egg. The antibodies are atsufficient titer to be protective against infection by CIAV of theprogeny of immunized hens. Thus, the present CIAV vaccine preventsclinical disease in the progeny of immunized chickens by preventing CIAVinfection in the chicks of immunized hens.

In the immunization method of the invention, the vaccine is administeredto chickens prior to the onset of egg production. For example, a validtime range for most if not all types of chickens is from about 4 toabout12 weeks of age. The lower time is relevant based on theage-resistance phenomenon noted with CIAV. Although the exact age candiffer among the different types of chickens, in the chicken strainstested resistance is present at as young as about 4 weeks of age. It isrecognized that in chickens that develop resistance at an earlier age,the vaccine can successfully be administered before 4 weeks (i.e. anytime after resistance develops). Similarly, for chicken types thatdevelop resistance later, the vaccine can successfully be administeredany time after resistance develops. Since resistance to CIAV disease canbe routinely determined, for example, by using the methods shown in theExamples, this parameter is routinely adjustable, such that theinvention is not limited to a particular lower age limit forimmunization.

The upper time limit is relevant based on two general considerations: 1)the need to immunize sufficiently in advance of the onset of eggproduction to allow antibody titers to develop in the immunized hen; and2) the need to immunize sufficiently in advance of the onset of eggproduction to allow clearance of the CIAV from the immunized hen. Theage of onset of egg production varies among the different types ofchickens. Thus, while 24 weeks is the approximate time of onset in thechickens tested, this parameter is not limited to that particular age,but is based on the routinely determinable age of onset for a givenpopulation of chickens.

In terms of the development of sufficient antibody titer, this isexpected to vary within routinely determinable parameters from chickento chicken. Thus, while 6 weeks prior to the onset of egg production hasbeen determined to be sufficient in the strains tested, the contemplatedtime frame encompasses any time that can be determined to be sufficientfor antibody production, including about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24 weeks (andintervening days) in advance of egg production. Methods of measuringantibody titer and determining sufficiency for protective immunizationof progeny are routine and are provided in the Examples herein.

In terms of the time needed to clear the virus prior to egg production,this is expected to vary within routinely determinable parameters fromchicken to chicken. For the chickens exemplified herein, the it wasdetermined that 12 weeks prior to egg productions is sufficient to clearthe virus. Because this parameter is also routinely measured, the timeframe contemplated encompasses any time sufficient to clear the virus,including about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 23, 24 weeks (and intervening days) in advance ofegg production. Methods of measuring virus titer and determiningclearance of the virus are routine and are provided in the Examplesherein.

It should also be noted that the upper and lower time limits foradministration of the vaccine are not necessarily based on the eggproduction status, antibody titer or virus titer of an individualchicken. Rather, it is the overall status of the group (e.g.,population, strain, etc.) of chickens to be immunized that is relevant.Thus, if a sufficient percentage of individual chickens within a groupare known or are expected (e.g., based on prior knowledge of the group)to be at the appropriate age for immunization, the immunization isconsidered successful.

The CIAV vaccine of the invention can be administered using any of thetypical methods. For example, an advantageous method is to administerthe vaccine in drinking water. The key features of the present wateradministered CIAV vaccine are: 1) The CIAV is apathogenic for the hostand is sufficiently invasive (at an acceptable input) to induce anadequate level of antibody.

2) The CIAV was demonstrated to spread.

3) The antibody induced will prevent the vertical transmission of achallenge virus.

4) The maternal antibody is efficiently transferred to the progeny andis protective.

5) The antibody will endure for an extended period of time.

The present data, strongly support the premise that the CIAV possessesthese key features.

The vaccine can, alternatively, be administered by parenterally,including by injection or by aerosol spray (e.g., of any mucousmembrane: nasal, pharyngeal, oral, ocular, intratracheal, cloacal, etc).

The invention provides a method of making a CIAV vaccine in an oncogeniccell line comprising subjecting the cell-cultured virus to more than onecycle of freezing and thawing, followed by maintaining the cells forabout 3 days at about 37° C., whereby contaminating virus from the cellline is killed. There are numerous oncogenic cell lines that have growthcharacteristics and other characteristics that make them advantageousfor growing CIAV. However, due to the existence in some of these celllines of contaminating viruses (e.g., the tumor virus associate with thetumor from which the cell line was isolated), using them to produce alive CIAV vaccine has been problematic. The invention addresses thisproblem by providing methods of inactivating the contaminating viruswithout killing the CIAV. These methods are described in the Examplesand elsewhere herein. Thus, the invention also provides a CIAV vaccine,comprising live CIAV passaged in an oncogenic cell line, wherein thevaccine does not cause Marek's Disease.

Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

EXAMPLES Example 1 Steps in Making the CIAV Vaccine in MSB-1 Cells

MSB-1 cells are maintained in vials frozen in liquid nitrogen until suchtime they are needed to expand into significant number for thepropagation of the CIAV.

MSB-1 cells are planted as described in the scientific literature intovarious tissue culture vessels in RPMI-1640 media supplemented withfetal calf serum. Cells are incubated at about 41° C. These cells growrapidly and can be frequently expanded to maintain actively growingcells.

The vaccine is produced by adding the CIAV virus to cells that have beenexpanded into new media such that the cell density is approximately 1 to5×10⁵ cells/ml media, and the virus input is at least about 1×10⁵TCID₅₀/ml media.

The virus-infected cells are incubated at about 41° C. for 4 to 7 days.Cells are microscopically examined for evidence of cell death as thedetermination of harvest time.

A step must be added to the virus harvest procedure to ensureinactivation of any residual Marek's disease virus that may be in theMSB-1 cells or that may be cell free. A proven effective procedure isthe filtering of the cells and media through a Pall 4.5 to 5 microncartridge to remove the MSB-1 cells followed by temperature treatment ofthe virus for about three days at about 37° C. to ensure inactivation ofcell-free Marek's disease virus. Alternatively, the virus may be frozenand thawed three times to sufficiently rupture the MSB-1 cells torelease and inactivate Marek's disease virus (an obligate intracellularpathogen). Then the virus fluid is subjected to a temperature treatmentof about 37° C. for 3 days to ensure complete inactivation of anyresidual Marek's disease virus.

Since the CIAV is very stable the vaccine can be supplied in a frozenform or in liquid form keptat refrigerated temperature of 2-7° C., orthe virus may be freeze dried.

Example 2 PCR and Restriction Analysis

Preparation of Intervet CIAV vaccine sample in MSB-1 cells. Due to theincompatibility of the blue dye contained in the Intervet CIAV chickenembryo-adapted and attenuated vaccine sample (Intervet CIAV) and the PCRtest, the sample was passed once in MSB-1 cells. MSB-1 cells wereinoculated with 1:2 and 1:10 dilutions of virus, and cells wereincubated for 96 hours prior to harvest. The culture media stillappeared blue due to the dye in the vaccine sample so the cells wereseparated from the supernatant by centrifugation and the cells werewashed twice with PBS. Both supernatant and cells were stored at −70 C.

PCR. CIAV PCR following the protocol of the Center for VeterinaryBiologics Laboratory (CVBL) in Ames, IA was conducted on the followingsamples:

-   -   1) CIAV, Del Ros strain    -   2) Intervet embryo-adapted commercial CIAV vaccine (Intervet        CIAV), serial no. 2448003    -   3) MSB-1 cells of passage 1 (P1) of Intervet CIAV passaged at a        1:2 dilution    -   4) Supernatant of P1 passaged at a 1:2 dilution    -   5) MSB-1 cells of passage 1 (P1) of Intervet CIAV passaged at a        1:10 dilution    -   6) Supernatant P1 passaged a 1:10 dilution    -   7) MSB-1 cells only        The primers are: 5′CTA/AGA/TCT/GCA/ACT/GCG/GA 3′ and        5′CCT/TGG/AAG/CGG/ATA/GTC/AT 3′

Restriction enzyme analysis. Part of the CVBL protocol to further verifyCAV, uses restriction enzyme analysis with HindIII, which states thatthe PCR product is cut one time. For restriction enzyme analysis, thePCR products were cut out of the agarose gel and the DNA was purified.Then the products from the cell samples were combined with thesupernatant samples before cutting with HindIII. TABLE 1 Results. PCRamplification and restriction enzyme analysis. PCR positive/negativeHindIII Sample (bp) fragments CIAV, Del Ros strain positive (419 bp) 281and 138 bp Intervet CIAV positive (419 bp) 419 bp 1:2 dilution of P1 -cells positive (419 bp) 419 bp 1:2 dilution of P1 - supernatant positive(419 bp) 1:10 dilution of P1 - cells positive (419 bp) 419 bp 1:10dilution of P1 - supernatant positive (419 bp) MSB-1 cells only NegativeN/A

Discussion. The primers used by CVBL were designed to the Cuxhaven-1isolate which amplifies a 419bp region starting at nucleotide 654 andends at nucleotide 1072 of the genomic DNA-plus strand. This regionoverlaps 3 ORF's of which one encodes for VP-1, capsid protein. Theseprimers amplified the sample. Surprisingly, the restriction enzyme thatnormally cuts the PCR product did not cut this sample. This means thatthe sample is probably CAV due to amplification by the primers, but itis different from the Del Ros (Delaware), Cl-1 (Maryland), Cuxhaven-1(Germany), and the Gifu-1 isolate (Japan). The difference in thenucleotide sequence may be just one base change at the HindIII site suchthat the enzyme's recognition site has been altered. The difference mayalso be due to many base changes, but DNA sequencing of the PCR productwould be needed to determine the similarity between the Del Ros strainand the sample.

Example 3 Results of CIAV-DR Bird Studies

Pathogenicity Evaluation of the CIAV, Del-Ros Strain (CIAV-DR):

1) 2-day-old, CAV-negative SPF chicks; 20 inoculates, 10 negativecontrols; 10^(6.9) TCID₅₀ of CIAV-DR in 0.2 ml; per os.

2) The clinical and serological results were as follows: Week p.i/% Dayp.i. (dpi) Day p.i./ Weight Reduction Hemat. Val. ELISA (total) Treat. 12 3 14 21 28 % Mort. % Gross Les. 28 35 Negative 0 0 0 39 35 36^(a) 40(NS^(b)) 0 1/6 0/6 Control CAV 0 9 0 32 31 34 0 30 19/20 20/20 Del-Ros^(a)Mean hematocrit values^(b)Non-specific

This study demonstrated that the Del-Ros strain is of low virulencebecause of the fact that it had little or no impact on growth rate,anemia, mortality and gross lesions when administered to the mostsusceptible age, CIAV-negative chickens by a natural route (i.e., oral).However, Del-Ros strain was sufficiently invasive to induce a goodantibody response (i.e., 100% ELISA positive; VN titers ranging from1:256-1:1024. The gross lesions observed were restricted to hemorrhagesof muscles and pale bone marrow.

Pathogenicity Evaluation of 3 Strains of CIAV; Del-Ros, CAV-9 and Texas:

1) 2-day-old, CAV-negative SPF chicks; 10 chicks per virus strain, 5negative controls; approx. 105.7 TCID50 of virus in 0.2 ml; IA.

2) The clinical and serological results are as follows: Week p.i/% Dayp.i./ Day p.i./ Weight Reduction Hemat. Val. % % ELISA (total) Treat. 12 3 14 21 28 Mort. Gross Les. 28 Control 0 0 0 37 33  33* 0 0 0/5Del-Ros 0 9 0 32 31 34 0 30  9/10 CAV-9 32 0 4 29 28 30 50 70 5/5 Texas29 0 1 24 25 34 70 70 3/3*Mean hematocrit valuesThis study demonstrated that the Texas strain of CIAV was sufficientlyvirulent to be used as a challenge virus when inoculated into 1- or2-day-old susceptible chicks by a parenteral route (e.g.,intra-abdominal). The gross lesions observed included; thymic atrophy,subcutaneous and intramuscular hemorrhaging, pale bone marrow, enlargedend congested liver lobes and gangrenous dermatitis.

Example 4 A Study Conducted with Chicken Infectious Anemia Virus, DELROS Strain, by Serial Back Passaging in SPF Chickens to DemonstrateVirus Does Not Become Virulent Introduction

A host animal reversion to virulence study was conducted on the chickeninfectious anemia virus, Del Ros strain (CIAV-DR) by serial backpassagein CIAV serologically negative SPF chickens.

Procedure

The potential reversion to virulence of the CIAV-DR live vaccine byserial backpassage in the host animal was evaluated by dailyobservations for clinical signs, hematocrit value determinations andpostmortem examinations for gross lesions characteristic of CIA.

Chickens used in the reversion to virulence study were CIAV-negative,SPF leghorn-type purchased from SPAFAS, Storrs CT. Three-week-oldchickens were delivered banded for identification and at that time allwere bled for CIAV serology to determine the CIAV serological status(ELISA; IDEXX CAV Kit) of the birds. At four weeks of age, eight tothirteen (backpassages 2-4) or twenty-four to twenty-eight (backpassages1 and 5) chickens per virus backpassage were vaccinated with a 10 μldose (10^(5.8) TCID₅₀, 1^(st) backpassage; a 20% suspension of a pooledtissue homogenate from the preceding backpassage given at a rate of 10μl/bird, 2nd through 5th backpassage) via the wing web route. Thisseries of five backpassages occurred over an eight-week period.

Liver, spleen and thymus were removed from eight euthanized chickens perbackpassage at seven days post vaccination (DPV) to prepare a 20%suspension of a pooled tissue homogenate (Waring Blender) in RPMI 1640medium containing antibiotics, but no serum and used as working stock inthe inoculation of chickens for backpassage and virus isolation in MSB-1cells according to the procedure of Yuasa et al. [Natl. Inst. Anim.Health Q (Tokyo) 23:75-77,1983].

All of the chickens of each backpassage were observed daily for clinicalsigns for seven (backpassages 2-4) or twenty-one DPV and the findingsrecorded. Blood was collected from all remaining chickens in backpassageone and five at fourteen and twenty-one DPV for hematocrit valuedetermination. Chickens euthanized at seven and twenty-one DPV wereexamined for gross lesions characteristic of CIA.

An analysis of phenotypic stability was conducted on the virus recoveredform the fifth backpassage in chickens as compared by standard indirectfluorescent antibody assay (IFA).

Results

The results obtained reveal that the CIAV-DR did not induce morbidityand mortality, anemia and gross lesions characteristic of CIA whensubjected to five serial backpassages in chickens. Additionally, it wasdemonstrated that the CIAV remained phenotypically stable in theprocess.

Results of pre-trial blood samples for CIAV serological status, virusrecovery from tissue homogenate extracts and post-mortem and hematocritvalue findings at seven, fourteen and twenty-one DPV for the fivebackpassages are given in tables 1-5.

A summary of the virus recovery, hematocrit value and post-mortemexamination results are given in table 6.

Summary

This reversion to virulence study conducted with a live CIAV-DR,administered by wing web to four week old chickens, demonstrated thatthe virus did not revert to virulence when subjected to five serialbackpassages, based on clinical observations and postmortemexaminations. TABLE 1 ELISA, Virus Recovery, Hematocrit and Post-mortemResults for the First Serial Backpassage. Hematocrit Bird No. Band No.ELISA S/N 14 d/21 d CIAV SGL*** 1 1  1.09*  35/30** None 2 2 1.15 29/31None 3 3 1.13 30/30 None 4 4 1.16 30/33 None 5 5 1.25 NA/NA None 6 61.24 35/36 None 7 7 1.31 NA/NA None 8 8 0.91 NA/NA None 9 9 0.77 34/29None 10 10 1.06 30/31 None 11 11 1.14 34/32 None 12 12 1.25 30/31 None13 14 1.32 NA/NA None 14 15 1.13 34/37 None 15 16 0.95 31/27 None 16 171.08 NA/NA None 17 18 1.14 32/30 None 18 19 1.2  32/34 None 19 20 1.3 34/31 None 20 21 1.35 NA/NA None 21 22 1.41 NA/NA None 22 23 0.96 NA/NANone 23 25 1.1  30/28 None 24 26 1.18 34/32 None 25 27 1.29 32/32 None26 29 1.39 29/30 None 27 30 1.38 35/34 None 28 31 1.04 32/33 NoneVirus Recovery from a Pooled Tissue Homogenate = Positive*S/N Ratios >0.6 = Negative (IDEXX Kit Interpretation)**Hematocrit Value >25 = Negative***Specific Gross Lesions

TABLE 2 ELISA, Virus Recovery and Post-mortem Results for the SecondSerial Backpassage. Bird No. Band No. ELISA S/N CIAV SGL** 1 32 1.06*None 2 33 1.1 None 3 34 1.02 None 4 35 0.93 None 5 36 1.01 None 6 370.98 None 7 38 1.03 None 8 39 1 None 9 40 0.97 None 10 41 0.99 None 1142 1 None 12 43 0.96 None 13 44 0.93 NoneVirus Recovery from a Pooled Tissue Homogenate = Positive*S/N Ratios >0.6 = Negative (IDEXX Kit Interpretation)**Specific Gross Lesions

TABLE 3 ELISA, Virus Recovery and Post-mortem Results for the ThirdSerial Backpassage. Bird No. Band No. ELISA S/N CIAV SGL** 1 45 0.9*None 2 46 0.94 None 3 47 0.61 None 4 48 0.78 None 5 49 0.7 None 6 500.84 None 7 51 0.83 None 8 52 0.97 None 9 53 0.88 None 10 54 0.81 None11 55 0.78 None 12 56 0.83 None 13 57 0.85 NoneVirus Recovery from a Pooled Tissue Homogenate = Positive*S/N Ratio >0.6 = Negative (IDEXX Kit Interpretation)**Specific Gross Lesions

TABLE 4 ELISA, Virus Recovery and Post-mortem Results the Fourth SerialBackpassage. Bird No. Band No. ELISA S/N CIAV SGL** 1 59 0.93* None 2 600.9 None 3 61 0.86 None 4 62 0.9 None 5 63 0.88 None 6 64 0.87 None 7 670.83 None 8 70 0.95 NoneVirus Recovery from a Pooled Tissue Homogenate = Positive*S/N Ratio >0.6 = Negative (IDEXX Kit Interpretation)**Specific Gross Lesions

TABLE 6 Summary of Hematocrit, Virus Recovery and Post-mortem Results ofChickens. Back Virus Passage Hematocrit Recovery Post-Mortem 1  0/20*1/1** 0/28 2 — 1/1 0/13 3 — 1/1 0/13 4 — 1/1 0/8  5 0/16 1/1 0/24*Number Positive/Number in Group**Virus Recovery for a Pooled Tissue Homogenate

TABLE 5 ELISA, Virus Recovery, Hematocrit and Post-mortem Results forthe Fifth Serial Backpassage. Hematocrit Bird No. Band No. ELISA S/N 14d/21 d CIAV SGL*** 1 2 0.81* NA/NA None 2 3 0.61  32/34** None 3 4 0.7236/31 None 4 5 0.79 33/32 None 5 6 0.87 32/35 None 6 7 1.09 NA/NA None 79 0.7 34/35 None 8 10 0.79 NA/NA None 9 11 0.9 NA/NA None 10 13 0.9331/33 None 11 14 1.03 NA/NA None 12 15 0.97 32/35 None 13 18 0.8 26/30None 14 19 0.84 35/33 None 15 20 0.92 33/33 None 16 21 0.91 26/32 None17 23 1.05 29/35 None 18 24 0.61 NA/NA None 19 25 0.89 28/35 None 20 260.92 30/30 None 21 28 0.97 NA/NA None 22 29 0.96 33/35 None 23 30 0.9932/35 None 24 31 0.95 NA/NA NoneVirus Recovery from a Pooled Tissue Homogenate = Positive*S/N Ratio >0.6 = Negative (IDEXX Kit Interpretation)**Hematocrit Value >25 = Negative***Specific Gross Lesions

Example 5 Results of a Shed/Spread and Vertical Transmission StudyConducted in SPF Chickens Following Wing Web Administration of a LiveChicken Anemia Virus Vaccine Introduction

A host animal shed/spread and vertical transmission study was conductedin chicken infectious anemia virus (CIAV)-negative, SPF chickens on achicken infectious anemia virus, Del Ros strain, (CIAV-DR) administeredby the wing web route. To assess shed and spread of CIAV live vaccine tocontact controls, cloacal swabs were collected from vaccinated andcontact control chickens for a 4 week post vaccination (p.v.) period andassayed for virus isolation in MSB-1 cells. To evaluate verticaltransmission (i.e., p.v.) of CIAV live vaccine, pools of livers of19-day-old embryos derived from eggs laid by vaccinated hens wereassayed for virus by isolation in MSB-1 cells and by PCR detection.

Procedure

The methods used to determine the shed/spread and vertical transmissionof a new CIA master seed virus were conducted in CIAV-negative, SPFchickens vaccinated at 12 weeks of age. The possible shed and spread ofwing web administered CIAV vaccine (live virus) was evaluated bycollecting cloacal swabs from vaccinated and contact control chickensfor a 4 week p.v. period followed by virus isolation attempts in MSB-1cells. The possibility of vertical transmission of live CIAV vaccine wasexamined by assaying pools of livers of 19-day-old embryos derived fromall of the fertile eggs laid by all of the vaccinated hens for virus byisolation in MSB-1 cells and by PCR detection. Livers of embryos from 3settings of eggs from negative control hens were evaluated in the samemanner.

Chickens used in the shed/spread and vertical transmission study wereCIAV-negative, SPF leghorn-type (SPF flock L103) purchased from SPAFAS.Birds were banded for identification. Ten randomly selected chickens at12 weeks of age were bled for CIAV serology to confirm the negativestatus (ELISA; IDEXX CAV Kit) of the birds. On the same day,thirty-seven chickens (30 females and 7 males) were vaccinated with a 10μl dose (10⁴³ TCID₅₀) of the live CIAV vaccine by the wing web route.Fifteen females (same source and hatch) were intermixed with thevaccinates as contact controls. Negative control chickens from the samesource and hatch were maintained. Chickens of both groups were observeddaily for morbidity and mortality and findings recorded for the durationof the study period.

Cloacal swab collections from fifteen randomly selected vaccinatedchickens and the fifteen contact controls were made at 3-7 day intervalsfor a 4 week p.v. period. Cloacal swabs were pooled for virusreisolation by combining 3 groups of 5 swabs per treatment per samplingtime. Virus recovery attempts were made in MSB-1 cells according to theprocedure of Yuasa et al. [Natl. Inst. Anim. Health Q (Tokyo) 23:75-77,1983].

Livers were aseptically collected from live and dead embryos (derivedfrom fertile eggs laid by vaccinated and negative control hens for a 3week p.v. period) at 19 days of incubation and packaged/stored (−20° C.)in pools of 3-6 livers for future processing. Twenty percent (w/v) liver(pools) suspensions were prepared in RPMI 1640 medium plus 5% FBS forvirus reisolation in MSB-1 cells according to the procedure of Yuasa etal. [Natl. Inst. Anim. Health Q (Tokyo) 23:75-77, 1983]. Prior toinitiating a CIAV isolation procedure on test hens, an assessment of thesensitivity of the CIAV isolation method outlined in the “shed/spreadand vertical transmission protocol” was conducted. Briefly, thisprocedure entailed harvesting livers from CIAV-antibody free SPF embryosat 19 days of incubation and preparing four pools of five livers each.One liver pool was maintained as a negative control; second, third andfourth pools were inoculated with 10, 100 and 1000 TCID₅₀ of CIAV pergram of tissue, respectively.

In addition to virus reisolation assays conducted, attempts to detectCIAV by PCR according to the procedure of Taylor and Ryncarz (Center forVeterinary Biologics Laboratory, NVSL, VS, APHIS, USDA, Ames, Iowa) wereundertaken.

Results

The results revealed that 10^(4.3) TCID₅₀ of the CIAV-DR administered tobreeders at 12 weeks of age via the wing web is shed for as much as 21days and that it will spread to contact controls. However, the virus wasnot vertically transmitted by breeders to their progeny as demonstratedby virus reisolation and PCR assays. The breeders did not exhibit anyadverse clinical effects from the vaccine administration.

Results of ELISA on pre-trial blood samples confirmed that the chickensused in this study were CIAV-antibody negative (table 1).

Results of virus reisolation attempts on cloacal swab pools ofvaccinates and contact controls are recorded in table 2. These data showthat CIAV was being shed by vaccinates as soon as 3 days p.v. and thisshed continued through 21 days p.v., but not 28 days p.v. Additionally,the data show that the shed CIAV readily spread to the contact controlswho also shed the virus for similar period of time.

A summary of the virus reisolation and PCR detection attempts on embryoliver suspensions derived from the fertile eggs of vaccinates andnegative controls are given in table 3. These data reveal that CIAVcould not be isolated from embryo liver suspensions of negative controland vaccinates by passage in MSB-1 cells or be detected by PCR. Theresults of a CIAV isolation sensitivity assessment in MSB-1 cellsdemonstrated that varying levels of CIAV (i.e., 10-1000 TCID₅₀/gram oftissue) was detected by this method following several cell culturepassages (table 4). There was complete correlation in the resultsobtained using these two methods on test samples.

Summary

This shed/spread and vertical transmission study was based on an effortto isolate and/or detect live CIAV in cloacal swabs and fertile eggs(i.e., embryo liver suspensions) collected from wing web vaccinated(10^(4.3) TCID₅₀/dose) and negative control hens. The resultsdemonstrated that the virus was shed and spread for a limited period oftime (21 dpv) but that this virus was not transmitted vertically whenadministered at 12 weeks of age. TABLE 1 Pre-Trial Blood Sample ELISAResults. Bird No. Band No. S/N Ratio CIAV Serol. Status 1 554 0.91Neg^(a) 2 557 0.93 Neg. 3 565 0.92 Neg. 4 566 0.96 Neg. 5 574 0.96 Neg.6 579 1 Neg. 7 584 1 Neg. 8 585 1 Neg. 9 731 0.99 Neg. 10 740 0.99 Neg.^(a)Negative = S/N Ratio >0.6 (IDEXX Kit Interpretation)

TABLE 2 Shed/Spread: Summary of Virus Reisolation from Cloacal SwabPools of Vaccinated and Contact Control Chickens. Vaccinate ContactControl Cloacal Cloacal Swab Pools Cloacal Swab Pools Swab (dpv)^(a) 1 23 1 2 3 3 N^(b) P^(c) N N N P 7 P P P P P P 12 N P N N P N 16 P P N N NP 21 P P P P N N 28 N N N N N N^(a)Cloacal Swab Collection (Days Post Vaccination).^(b)Negative^(c)Positive = Characteristic CIAV CPE Observed

TABLE 3 Vertical Transmission: Summary of Virus Reisolation and PCRDetection Assays on Embryo Liver Suspensions Derived from the FertileEggs of Vaccinates and Negative Controls. Treatment Virus ReisolationPCR Detection 1^(a)  0/12^(b) 0/12 2a^(c) 0/17 0/17 2b 0/15 0/15 2c 0/190/19 2d 0/18 0/18 Pos. Con.^(d) 6/6  5/6  Neg. Con.^(e) 0/6  0/6 ^(a)SPAFAS Negative Controls^(b)Number Positive/Total Tested^(c)Vaccinates - four groups (2a-2d)^(d)Positive Controls (MSB-1 Propagated Del-Ros and Texas Strains ofCIAV)^(e)Negative Controls (MSB-1 cells and/or Reagent Mix)

TABLE 4 Results of a CIAV Isolation Sensitivity Assessment. MSB-1Passages Treatment 1 2 3 4 5 6 10 TC1D₅₀ ^(a)  0/5^(b) 0/5 0/5 0/5 0/55/5 100 TC1D₅₀ 0/5 0/5 0/5 0/5 0/5 5/5 1000 TC1D₅₀ 0/5 0/5 0/5 0/5 3/55/5 Uninf. Cont.^(c) 0/5 0/5 0/5 0/5 0/5 0/5^(a)TC1D₅₀/Gram of Tissue^(b)Number Positive (Characteristic CIAV CPE Observed)/Total^(c)Uninfected Controls

Example 6 Efficacy Study Conducted in Progeny of SPF Chickens 34 and 49Weeks Following Wing Web Administration of a Live Chicken Anemia VirusVaccine Introduction

A study to evaluate vaccine efficacy and duration of immunity (DOI) at34 and 49 weeks post wing web vaccination was conducted by challengingday-old progeny of hens vaccinated with a chicken infectious anemiavirus, Del Ros strain, (CIAV-DR). The study assessed CIAV maternalantibody protection (passive immunity) provided to chicks against achallenge with virulent CIAV.

Procedure

Efficacy and duration of immunity of the were conducted in the progenyof CIAV-negative, SPF chickens vaccinated at 9 weeks of age with CIAVvaccine administered via the wing web route. Duration of immunity wasevaluated by challenging progeny, hatched from fertile eggs laid by hensat 34 and 49 weeks post vaccination, followed by observations forclinical signs, hematocrit value determinations and post-mortemexaminations for gross lesions characteristic of CIA.

Chickens used in this study were CIAV-negative, SPF leghorn-typepurchased from SPAFAS. Birds were wing-banded for identification. Tenrandomly selected chickens at 9-weeks-of-age were bled for CIAV serologyto confirm the negative serological status (ELISA, IDEXX CAV Kit) of thebirds. On the same day, 70 chickens (60 females and 10 males) werevaccinated with a 10 μl dose (10^(4.2) TCID₅₀) of the live CIAV vaccineby the wing web route. Negative control chickens from the same sourceand hatch were maintained. The dose was determined as the average of 5replicate titers conducted immediately after vaccination. Chickens ofboth groups were observed daily for morbidity and mortality and thefindings recorded for the duration of the study period.

A one-week collection of eggs from 52 vaccinated hens (43-weeks-of-age)were used to evaluate progeny of breeders at 34 weeks post CIAVvaccination (DOI Test 2). A second one-week collection of eggs from 48vaccinated hens (58 weeks of age to assess progeny of breeders at 49weeks post CIAV vaccination (DOI Test 3).

Forty-day-old chicks, each from CIAV vaccinated and non-vaccinatedbreeders, were challenged with liver homogenate extract derived fromchicks inoculated with a Texas field isolate of CIAV. Each chick wasinoculated intra-abdominally with approximately 10^(2.6) CID₅₀ per 0.2ml. Negative control groups consisted of 25 chicks.

Chicks of all treatment groups were maintained in separate filtered-air,negative-pressure isolators and observed daily for depression, ruffledfeathers and mortality. Blood samples were collected from all of thechicks at 14 and 21-22 days post challenge for hematocrit valuedeterminations as a measure of anemia. The procedure used fordetermining hematocrit values was that of Rosenberger and Cloud (AvianDis. 33:753-759, 1989). Additionally, chicks of all treatment groupswere examined for gross lesions characteristic of CIA (i.e., pale bonemarrow, swelling and discoloration of the liver and spleen andhemorrhagic lesions in the skin and muscles) at 21-22 days postchallenge. Treatment comparisons were based on the number of individualswithin a treatment (per total examined) exhibiting specific grosslesions of CIA.

Results

The results of the two DOI challenge tests, reported herein,demonstrated that 10^(4.2) TCID₅₀ of virus administered to breeders at 9weeks of age via the wing web protected progeny against morbidity andmortality, anemia and gross lesions characteristic of CIA through 49weeks post vaccination as determined by statistical evaluation.

Pre-study blood sample ELISA results were found to confirm theCIAV-negative status of the semi-mature chickens acquired from SPAFASfor use in this study and are presented in table 1.

Results of hematocrit value determinations, clinical-sign findings andpost-mortem examinations of CIAV challenged and non-challenged day-oldchicks are recorded in tables 2, 3 and 4 (DOI Test 2) and 8, 9 and 10(DOI Test 3); tables 5 and 11, respectively, summarize this information.Chicks with gross lesion scores ≧1, for any one of the tissues examined(i.e., liver, muscle, bone marrow and thymus), were recorded as CIApositive (tables 5 and 11). The death of chicks (table 2; derived fromCIAV vaccinated breeders) numbered 3, 8, 22, 26, 27 and 40 in DOI test 2resulted from suffocation in an isolator glove. Statistical evaluations(Fisher's Exact Probability Test; tables 6 and 12) of hematocrit valuesand clinical signs of Test 2 and 3 chicks revealed that progeny of CIAVvaccinated versus non vaccinated breeders were protected against anemiaand mortality at a statistically significant level (p<0.001) whenchallenged with a virulent field isolate of CIAV. A statisticallysignificant difference (p=0.027) in morbidity was demonstrated amongchallenged progeny in DOI Test 3. Statistical assessment (Mann-WhitneyTest; tables 7 and 13) of gross lesion scores revealed similar findingsas those reported above; i.e., a statistically significant differenceand in the bone marrow (p<0.001 and p=0.021, respectively) and thymus(p<0.001) gross lesion scores of progeny derived from vaccinated versusnon-vaccinated breeders. No significant differences were demonstratedfor liver and muscle lesions among challenged progeny.

Summary

This assessment of vaccine efficacy and immunity duration was based on aday-old, intra abdominal challenge of progeny derived from breedersvaccinated at 9 weeks of age with live CIAV-DR vaccine administered bythe wing web route. The results revealed that the CIAV vaccine inducedmaternal antibodies which protected chicks at a statisticallysignificant difference of p<0.05, against a virulent challenge with afield strain of CIAV, based on evidence of anemia at 14 and 21 days postchallenge, clinical signs and gross lesions of the bone marrow andthymus when compared to challenge control chicks. TABLE 1 Pre-TrialBlood Sample ELISA Results of 9 Week Old Chickens Prior to Vaccinationwith Wing Web Administered CIAV to Confirm Negative Serological Status.CIAV Serol. Bird No. Band No. S/N Ratio Status^(a) 1 602 0.88 Neg^(b) 2608 0.84 Neg 3 616 0.9 Neg 4 620 0.81 Neg 5 621 0.78 Neg 6 627 0.85 Neg7 631 0.87 Neg 8 634 0.82 Neg 9 644 0.85 Neg 10 661 0.7 Neg^(a)CIAV Serological Status^(b)Negative = S/N Ratio >0.6 (IDEXX Kit Interpretation)

TABLE 2 Test 2 Hematocrit Values, Clinical Signs and CIA Gross LesionScores of Chicks Challenged at 34 Weeks Following Wing Web AdministeredCIA Vaccine. Clin. Hematocrit Values Signs^(a) Gross Lesion Scores Bird14 Day 21 Day Mor./ Thy- Mus- No. pc^(b) pc Mort.^(c) Liver BM^(d) muscle 1 28 35 N/N  O^(e) 0 0 0 2 33 32 N/N 0 0 0 0 3 35 ND^(f) N/NCAM^(g)0 0 0 0 4 32 39 N/N 0 0 0 0 5 32 34 N/N 0 0 0 0 6 26 27 N/N 0 0 0 0 7 2832 N/N 0 0 0 0 8 32 ND N/NCAM 0 0 0 0 9 32 33 N/N 0 0 0 0 10 32 24^(h)N/N 0 2 1 1 11 26 12 P/N^(i) 0 2 2 0 12 33 26 N/N 0 2 2 0 13 27 31 N/N 00 0 0 14 32 35 N/N 0 0 0 0 15 33 32 N/N 0 0 0 0 16 60 39 N/N 0 0 0 0 1758 37 N/N 0 0 0 0 18 30 24 N/N 0 1 2 0 19 33 34 N/N 0 0 0 0 20 21 17N/N0 0 3 2 0 21 58 35 N/N 0 0 0 0 22 32 ND N/NCAM 0 0 0 0 23 33 37 N/N 02 1 0 24 34 36 N/N 0 0 0 0 25 29 33 N/N 0 0 0 0 26 30 ND^(f) N/NCAM^(g) O^(e) 0 0 0 27 34 ND N/NCAM 0 0 0 0 28 35 32 N/N 0 0 0 0 29 35 27 N/N 00 0 0 30 28 23^(h) N/N 0 1 2 0 31 30 31 N/N 0 0 0 0 32 ND ND N/P^(i) 0 00 0 33 33 35 N/N 0 0 0 0 34 34 41 N/N 0 0 0 0 35 27 36 N/N 0 0 0 0 36 3234 N/N 0 0 0 0 37 30 21 N/N 0 0 0 0 38 33 36 N/N 0 0 0 0 39 31 34 N/N 00 0 0 40 30 ND N/NCAM 0 0 0 0 Pos./Tot.^(j) 1/39 6/33 1/40/1/34 0/407/40 7/40 1/40^(a)Clinical Signs^(b)Post Challenge^(c)Morbidity (Depression and/or Ruffled Feathers)/Mortality^(d)Bone Marrow^(e)0 = Normal; 1 = Slight; 2 = Moderate; 3 = Severe Not Done^(f)Not None^(g)Negative/Non-CIAV Associated Mortality^(h)Hematocrit Values of ≦ 25 = Anemia^(i)Negative/Positive (CIAV Associated Mortality)^(j)Number Positive/Total

TABLE 3 Test 2 Hematocrit Values, Clinical Signs and CIA Gross LesionScores of Challenged Chicks from Non-Vaccinated Breeders. Clin.Hematocrit Values Signs^(a) Gross Lesion Scores Bird 14 Day 21 Day Mor./Thy- Mus- No. pc^(b) pc Mort.^(c) Liver BM^(d) mus cle 1 23^(e) ND^(f)N/P^(g)  0^(h) 2 3 1 2 18 ND N/P 0 2 2 0 3 29 22 N/N 0 0 0 0 4 26 20 P/N0 0 3 0 5 20 ND N/P 0 3 2 1 6 28 26 N/N 0  0− 0 0 7 21 57 N/N 0 0 3 0 820 ND N/P 0 3 3 0 9 21 21 N/N 0 2 2 0 10 18 ND N/P 0 3 3 2 11 32 24 N/N0 2 1 0 12 21 ND N/P 0 3 3 1 13 26 24 N/N 0 0 0 0 14 25 19 N/N 0 0 1 015 25 45 N/N 0 2 1 0 16 28 30 N/N 0 2 3 0 17 27 10 P/N 0 3 3 0 18 16 NDP/P 0 2 2 0 19 22 25 N/N 0 0 0 0 20 18 24 N/N 0 0 2 2 21 20 ND N/P 0 3 21 22 28 20 N/N 0 1 3 0 23 26 15 P/N 0 2 1 0 24 22 28 N/N 0 0 0 0 25 17ND P/P 2 3 3 2 26 24^(e) 30 N/N O^(h) 0 2 0 27 40 56 N/N 0 2 2 0 28 3015 N/N 0 2 2 0 29 29 29 N/N 0 0 0 0 30 31 27 N/N 0 1 2 0 31 25 32 N/N 01 2 0 32 25 13 P/N 0 3 3 0 33 21 27 N/N 0 0 0 0 34 28 21 N/N 0 2 2 0 3530 28 N/N 0 0 0 0 36 30 ND^(f) N/P^(g) 0 3 3 1 37 28 23 N/N 0 0 0 0 3870 13 N/N 0 2 1 0 39 23 25 N/N 0 0 1 0 40 25 27 N/N 0 0 0 0Pos./Tot.^(i) 22/40 17/30 6/40/10/40 1/40 24/40 30/40 8/40^(a)Clinical Signs^(b)Post Challenge^(c)Morbidity (Depression and/or Ruffled Feathers)/Mortality^(d)Bone Marrow^(e)Hematocrit Values ≦ 25 = Anemia^(f)Not None^(g)Negative/Positive (CIAV Associated Mortality)^(h)0 = Normal; 1 = Slight; 2 = Moderate; 3 = Severe^(i)Number Positive/Total

TABLE 4 Test 2 Hematocrit Values, Clinical Signs and CIA Gross LesionScores of Chicks from Non-Vaccinated Breeders; Not Challenged. Clin.Hematocrit Values Signs^(a) Gross Lesion Scores Bird 14 Day 21 Day Mor./Thy- Mus- No. pc^(b) pc Mort.^(c) Liver BM^(d) mus cle 1 37 38  N/N^(e) 0^(f) 0 0 0 2 38 35 N/N 0 0 0 0 3 35 30 N/N 0 0 0 0 4 40 35 N/N 0 0 0 05 36 37 N/N 0 0 0 0 6 38 36 N/N 0 0 0 0 7 35 36 N/N 0 0 0 0 8 28 38 N/N0 0 0 0 9 NS^(g) 35 N/N 0 0 0 0 10 31 NS N/N 0 0 0 0 11 36 36 N/N 0 0 00 12 37 35 N/N 0 0 0 0 13 36 33 N/N 0 0 0 0 14 31 42 N/N 0 0 0 0 15 3940 N/N 0 0 0 0 16 35 37 N/N 0 0 0 0 17 40 36 N/N 0 0 0 0 18 35 33 N/N 00 0 0 19 32 35 N/N 0 0 0 0 20 33 35 N/N 0 0 0 0 21 30 45 N/N 0 0 0 0 2239 39 N/N 0 0 0 0 23 34 40 N/N 0 0 0 0 24 33 38 N/N 0 0 0 0 25 35 27 N/N0 0 0 0 Pos./Tot.^(h) 0/24 0/24 0/25/0/25 0/25 0/25 0/25 0/25^(a)Clinical Signs^(b)Post Challenge^(c)Morbidity (Depression and/or Ruffled Feathers)/Mortality^(d)Bone Marrow^(e)Negative/Negative^(f)0 = Normal; 1 = Slight; 2 = Moderate; 3 = Severe^(g)No Sample^(h)Number Positive/Total

TABLE 5 Summary of Test 2 Hematocrit, Morbidity, Mortality and CIA GrossLesion Scores of Challenged and Non-Challenged Chicks. Test GroupHematocrit Morbidity Mortality PM^(a) CIAV Vaccinated^(b) 6/39 (15%)^(c)1/40 (3%) 1/34 (3%) 7/40 (18%)^(d) Non-Vaccinated^(b) 33/40 (83%) 6/40(15%) 10/40 (25%) 30/40 (75%) Negative Control 0/25 0/25 0/25 0/25^(a)Post-Mortem CIA Gross Lesion Scores^(b)Challenge Group^(c)Number Chicks Positive/Total^(d)Positive Chicks = Gross Lesion Scores ≧1

TABLE 6 Statistical Evaluation of Test 2 Hematocrit Values and CIAClinical Signs of Challenged Chicks using Fisher's Exact ProbabilityTest. Hematocrit Values Clinical Signs 14 Day 21 Day Morbid- Mortal-Com- Test Group pc^(a) pc ity ity bined CIAV 1/39 6′33 1/40 1/346/40^(b) Vaccinated Non- 22/40  17/30 6/40 10/40  34/40   Vaccinated pvalue <0.001 0.002 0.054 0.007 <0.001^(a)Post Challenge^(b)Combined Hematocrit Values and Clinical Signs

TABLE 7 Statistical Evaluation of Test 2 CIA Gross Lesion Scores ofChallenged Chicks from Vaccinated and Non-Vaccinated Breeders using theMann-Whitney Test Gross Lesion Scores^(a) Liver BM^(b) Thymus MuscleCombined^(c) p value 0.847 <0.001 <0.001 0.173 <0.001^(a)Raw Data Found in Tables 2 and 3^(b)Bone Marrow^(c)Combined Gross Lesion Scores

TABLE 8 Test 3 Hematocrit Values, Clinical Signs and CIA Gross LesionScores of Chicks Challenged at 49 Weeks Following Wing Web AdministeredCIA Vaccine. Hematocrit Values Clin. Scores Signs^(a) Gross Lesion Bird14 Day 21 Day Mor./ Thy- Mus- No. pc^(b) pc Mort.^(c) Liver BM^(d) muscle 1 31 45 N/N^(e) O^(f) 0 0 0 2 30 32 N/N 0 0 0 0 3 34 34 N/N 0 0 0 04 28 28 N/N 0 0 0 0 5 33 23^(g) N/N 0 0 0 0 6 32 30 N/N 0 0 0 0 7 24 36N/N 0 0 0 0 8 49 32 N/N 0 0 0 0 9 35 30 N/N 0 0 0 0 10 31 31 N/N 0 0 0 011 34 27 N/N 0 0 0 0 12 33 35 N/N 0 0 0 0 13 43 27 N/N 0 0 0 0 14 41 33N/N 0 0 0 0 15 25 30 N/N 0 0 0 0 16 35 31 N/N 0 0 0 0 17 30 32 N/N 0 0 00 18 32 35 N/N 0 0 0 0 19 30 33 N/N 0 0 0 0 20 32 28 N/N 0 0 0 0 21 3332 N/N 0 0 0 0 22 34 33 N/N 0 0 0 0 23 29 30 N/N 0 0 0 0 24 30 27 N/N 00 2 0 25 29 30 N/N 0 0 0 0 26 30 28 N/N^(e) 0^(f) 0 0 0 27 52 30 N/N 0 00 0 28 35 35 N/N 0 0 0 0 29 30 27 N/N 0 0 0 0 30 50 26 N/N 0 0 1 0 31 3531 N/N 0 0 0 0 32 35 34 N/N 0 0 0 0 33 20^(g) 30 N/N 0 0 0 0 34 31 30N/N 0 0 0 0 35 32 28 N/N 0 0 0 0 36 30 37 N/N 0 0 0 0 37 35 38 N/N 0 0 00 38 34 32 N/N 0 0 0 0 39 35 30 N/N 0 0 0 0 40 31 32 N/N 0 0 0 0Pos./Tot.^(h) 3/40 1/40 0/40/0/40 0/40 0/40 2/40 0/40^(a)Clinical Signs^(b)Post Challenge^(c)Morbidity (Depression and/or Ruffled Feathers)/Mortality^(d)Bone Marrow^(e)Negative/Negative^(f)0 = Normal; 1 = Slight; 2 = Moderate; 3 = Severe^(g)Hematocrit Values of ≦ 25 = Anemia^(h)Number Positive/Total

TABLE 9 Test 3 Hematocrit Values, Clinical Signs and CIA Gross LesionScores of Challenged Chicks from Non-Vaccinated Breeders. Clin.Hematocrit Values Signs^(a) Gross Lesion Scores Bird 14 Day 21 Day Mor./Thy- Mus- No. pc^(b) pc Mort.^(c) Liver BM^(d) mus cle 1 19^(e) ND^(f)N/P^(g) 2^(h) 2 3 2 2 22 32 N/N 0 0 0 0 3 50 ND P/P 0 0 3 0 4 32 28 N/N0 0 2 0 5 31 27 N/N 0 2 0 0 6 32 29 N/N 0 0 0 0 7 26 19 N/N 0 0 2 0 8 3027 N/N 0 0 0 0 9 23 ND P/P 0 2 2 0 10 17 29 N/N 0 0 0 0 11 23 35 N/N 0 00 0 12 20 ND N/P 0 3 3 0 13 18 ND P/P 0 0 2 0 14 22 ND N/P 0 2 3 1 15 4413 N/N 0 2 2 0 16 30 32 N/N 0 0 1 0 17 14 ND N/P 0 0 3 0 18 31 26 N/N 00 1 0 19 20 ND N/P 0 2 3 0 20 23 10 N/N 0 2 2 0 21 33 20 N/N 0 0 2 0 2223 ND P/P 0 0 3 0 23 22 ND N/P 0 0 3 0 24 29 27 N/N 0 2 2 0 25 30 15 N/N0 0 1 0 26 29 35 N/I N 0^(h) 0 0 0 27 24^(e) 33 N/N 0 0 0 0 28 27 20 N/N0 0 0 0 29 32 19 N/N 0 2 1 0 30 25 ND^(f) P/P 2 0 3 1 31 22 18 N/N 0 1 20 32 33 34 N/N 0 0 0 0 33 23 ND N/P^(g) 0 2 3 0 34 25 35 N/N 0 0 0 0 3516 ND N/P 0 0 3 0 36 28 15 N/N 0 0 0 0 37 29 25 N/N 0 0 0 0 38 30 32 N/N0 0 0 0 39 29 25 N/N 0 0 2 0 40 31 23 N/N 0 0 0 0 Pos./Tot.^(i) 19/4012/27 5/40 13/40 2/40 12/40 25/40 3/40^(a)Clinical Signs^(b)Post Challenge^(c)Morbidity (Depression and/or Ruffled Feathers)/Mortality^(d)Bone Marrow^(e)Hematocrit Values ≦25 = Anemia^(f)Not Done^(g)Negative/Positive (CIAV Associated Mortality)^(h)0 = Normal; 1 = Slight; 2 = Moderate; 3 = Severe^(i)Number Positive/Total

TABLE 10 Test 3 Hematocrit Values, Clinical Signs and CIA Gross LesionScores of Chicks from Non-Vaccinated Breeders; Not Challenged. Clin.Hematocrit Values Signs^(a) Gross Lesion Scores Bird 14 Day 21 Day Mor./Thy- Mus- No. pc^(b) pc Mort.^(c) Liver BM^(d) mus cle 1 35 34 N/N^(e)0^(f) 0 0 0 2 39 35 N N 0 0 0 0 3 33 34 N/N 0 0 0 0 4 37 35 N/N 0 0 0 05 38 33 N/N 0 0 0 0 6 32 35 N/N 0 0 0 0 7 35 37 N/N 0 0 0 0 8 29 39 N/N0 0 0 0 9 35 36 N/N 0 0 0 0 10 32 37 N/N 0 0 0 0 11 33 38 N/N 0 0 0 0 1233 34 N/N 0 0 0 0 13 31 35 N/N 0 0 0 0 14 30 35 N/N 0 0 0 0 15 36 40 N/N0 0 0 0 16 30 39 N/N 0 0 0 0 17 30 38 N/N 0 0 0 0 18 30 36 N/N 0 0 0 019 40 35 N/N 0 0 0 0 20 35 35 N/N 0 0 0 0 21 35 35 N/N 0 0 0 0 22 35 33N/N 0 0 0 0 23 34 41 N/N 0 0 0 0 24 28 41 N/N 0 0 0 0 25 32 38 N/N 0 0 00 Pos./Tot.^(g) 0/25 0/25 0/25/0/25 0/25 0/25 0/25 0/25^(a)Clinical Signs^(b)Post Challenge^(c)Morbidity (Depression and/or Ruffled Feathers)/Mortality^(d)Bone Marow^(e)Negative/Negative^(f)0 = Normal; 1 = Slight; 2 = Moderate; 3 = Severe^(g)Number Positive/Total

TABLE 11 Summary of Test 3 Hematocrit, Morbidity, Mortality and GrossLesion of Challenged and Non-Challenged Chicks. Test Group HematocritMorbidity Mortality PM^(a) CIAV Vaccinated^(b) 4/40 (10%)^(c) 0/40 0/402/40 (5%)^(d) Non-Vaccinated^(b) 29/40 (73%) 5/40 (13%) 13/40 (33%)26/40 (65%) Negative Control 0/25 0/25 0/25 0/25^(a)Post-Mortem CIA Gross Lesion Scores^(b)Challenge Group^(c)Number Positive/Total^(d)Positive Chicks = Gross Lesion Scores ≧1

TABLE 12 Statistical Evaluation of Test 3 Hematocrit Values and ClinicalSigns of Challenged Chicks using Fisher's Exact Probability TestHematocrit Values Clinical Signs 14 Day 21 Day Morbid- Mortal- TestGroup pc^(a) dc ity ity Combined CIAV 3/40 1/40 0/40 0/40 4/40^(b)Vaccinated Non- 19/40  12/27  5/40 13/40  30/40   Vaccinated p value<0.001 <0.001 0.027 <0.001 <0.001^(a)Post Challenge^(b)Combined Hematocrit Values and Clinical Signs

TABLE 13 Statistical Evaluation of Test 3 CIA Gross lesion Scores ofChallenged Chicks from Vaccinated and Non-Vaccinated Breeders using theMann-Whitney Test Gross Lesion Scores^(a) Liver BM^(b) Thymus MuscleCombined^(c) p value 0.7 0.021 <0.001 0.5637 <0.001^(a)Raw Data Found in Tables 8 and 9^(b)Bone Marrow^(c)Combined Gross Lesion Scores

Example 7 Efficacy of a Chicken Anemia Virus Vaccine Evaluated byMaternal Antibody Protection of Progeny from Chickens 27 and 37 WeeksFollowing Drinking Water Administration the Vaccine Introduction

Host animal efficacy and duration of immunity studies were conducted inchickens by challenge of day-old progeny hatched from 27 and 37 week-oldhens, which were previously vaccinated with chicken infectious anemiavirus, Del Ros strain (CIAV-DR) vaccine at 9 weeks of age by drinkingwater. The challenge procedure of progeny and parameters of measurementof efficacy by maternal antibody protection (passive immunity) providedby hens vaccinated in the drinking water were the same as for chickenanemia virus vaccine administered by the wing web route (see Example 6).

Procedure

Progeny were hatched from fertile eggs laid 18 and 28 weeks postvaccination when hens were 27 and 37 weeks of age, respectively.Intra-abdominal challenge of day-old progeny was used to evaluatematernal antibody protection provided by CIAV-DR following drinkingwater vaccination of CIAV-negative SPF chickens at 9 weeks of age. Postchallenge observations of progeny through 21 days of age includedclinical signs, hematocrit value determinations and post-mortemexaminations for gross lesions characteristic of chicken infectiousanemia (CIA).

Chickens used for vaccination in this study were CIAV negative, SPFleghorn-type purchased from SPAFAS, Inc. Birds were wing banded foridentification upon arrival. Twenty randomly selected chickens at 9weeks of age were bled for CIAV serology to confirm negative serologicalstatus using the IDEXX ELISA CIAV kit. On the same day, 40 females and 5males designated as vaccinates were water starved and then permitted todrink water containing CIAV-DR vaccine. The average of five replicatetiters of the CIAV vaccine conducted after vaccination in MSB-1 cellsdetermined a dose contained 10^(5.5) TCID₅₀. Negative control breederchickens from the same source and hatch date were maintained. Twoefficacy/duration of immunity studies identified as Study 1 and Study 2were conducted on progeny from 27 and 37 week-old hens, respectively

Chicks were challenged at one day of age with CIAV. The challenge viruswas liver homogenate extract derived from chicks inoculated with a Texasfield isolate of CIAV. Each chick was inoculated intra-abdominally withapproximately 10^(2.6) CID₅₀ per 0.2 ml.

Each study consisted of a group of progeny from non-vaccinated hensmaintained as non-challenged negative controls, a group of CIAVchallenged progeny from non-vaccinated hens that served as positivecontrols, and a group of CIAV challenged progeny from vaccinated hens.Chicks of all treatment groups were maintained in filtered air, negativepressure isolation units and observed through 21 days for depression,ruffled feathers and mortality. Blood samples were collected from allchicks at 14 and 21 days post challenge (dpc) for hematocrit valuedeterminations as a measure of anemia. The procedure used fordetermining hematocrit values was that of Rosenberger and Cloud (AvianDis. 33:753-759, 1989). A chick with a hematocrit value of ≦25 wasconsidered to be anemic. Additionally, chicks of all treatments wereexamined at 21 dpc for gross lesions characteristic of CIA includingpale bone marrow, swelling and discoloration of the liver and spleen,and hemorrhage lesions in the skin and muscles. Treatment comparisonswere based on the number of individuals within a treatment (per totalexamined) exhibiting specific gross lesions of CIA. Data werestatistically analyzed using Fisher's Exact Probability Test andMann-Whitney Test.

Results

Serological pre-vaccination serum samples using the IDEXX ELISA kitconfirmed the CIAV negative status of the 9-week-old chickens acquiredfrom SPAFAS, Inc. that were used in this study. ELISA results are givenin Table 1.

Results of the two studies reported herein demonstrated that 10^(5.5)TCID₅₀ of CIAV-DR vaccine administered by drinking water to 9-week-oldpullets significantly protected progeny at p<0.05 through 37 weeks ofage (i.e. 28 weeks post vaccination) when compared to progeny fromnon-vaccinated hens. A gross lesion score ≧1 for any one of the tissuesexamined (i.e. liver, bone marrow, thymus and muscle) was recorded as aCIA positive chick. There was a significant difference at p<0.05 inprogeny of vaccinated hens compared to non-vaccinated hens in Study 1and Study 2 against morbidity and mortality, anemia, and gross lesionscharacteristic of CIA.

Results of Study 1

Forty day-old chicks from non-vaccinated breeders challenged with CIAVwere evaluated in this study as the positive control group. The death ofone of 25 chicks from the non-challenged negative control group occurredearly in the test period and could not be attributed to any specificcause. Twenty-four negative controls remained for evaluation. A poweroutage in the isolator holding 40 challenged chicks from the CIAVvaccinated hens at 3 dpc and resulted in the death of 15 of the 40chicks leaving 25 chicks of this treatment group for evaluation in thisstudy (See Table 4). One chick from the CIAV vaccinated group died at 5dpc. The chick had no gross lesions or clinical signs of CIAV.Therefore, mortality was ruled due to non-CIAV related causes.

The 24 non-challenged negative control chicks did not exhibit morbidity,mortality or gross lesions of CIA. One of 22 serum samples collectedfrom chicks at 21 dpc had a hematocrit value of 23, but the chick had noother characteristic sign of CIA. Results are given in Table 2.

The challenge procedure induced CIA in progeny from non-vaccinatedbreeders. Hematocrit values ≦25 at either 14 or 21 dpc were demonstratedin 36 of 40 (90%) positive control chicks. Morbidity was noted in 5 of40 (12.5%) chicks, whereas, mortality was experienced in 10 of 40 (25%)chicks. Gross lesions were evident in 33 of 40 (82.5%) chicks. Resultsare given in Table 3.

Statistical evaluations by Fisher's Exact Probability Test of hematocritvalues demonstrated that there was a significant difference at p<0.001against anemia, a significant difference at p=0.012 against combinedmorbidity and mortality, and a significant difference of p<0.001 in thenumber of birds with CIA gross lesion scores in progeny from vaccinatedbreeders compared to progeny from non-vaccinated breeders. Statisticalanalysis of gross lesion scores by Mann-Whitney Test demonstrated asignificant difference of p<0.001 in the bone marrow and the thymus.There was a significant difference at p<0.001 by Fisher's Exact Test ofthe number of birds with gross lesions of progeny from vaccinatedbreeders compared to progeny from non-vaccinated breeders. Results andstatistical evaluations given in Tables 4, 5, 6 and 7.

Results of Study 2

The groups of study 2 consisted of non-challenged negative controls fromnon-vaccinated hens (n=25), CIAV challenged controls from non-vaccinatedhens (n=40) and CIAV challenged progeny from 37-week-old CIAV vaccinatedbreeder hens (n=40). Throughout the 21-day test, negative controlchickens remained free of anemia as determined by hematocrit values,morbidity, mortality and gross lesion scores associated with CIA.Results are given in Table 8.

The CIAV positive control chicks exhibited lowered hematocrit values,clinical signs and gross lesions typical of CIA. Hematocrit values ≦25at either 14 or 21 dpc were demonstrated in 32 of 39 (82.1%) positivecontrol chicks. Morbidity was noted in 6 of 40 (15.0%) chicks, andmortality was experienced in 12 of 40 (30.0%) chicks. Gross lesions wereevident at post mortem in 24/40 (60.0%) of chicks. Results are given inTable 9.

Following CIAV challenge a significant difference at p<0.05 wasdemonstrated in progeny from CIAV vaccinated hens compared to progenyfrom non-vaccinated hens in hematocrit values at 14 and 21 dpc, inmorbidity and mortality, and in gross lesions scores. Fisher's ExactProbability Test of hematocrit values demonstrated a significantdifference at p<0.001 against anemia, a significant difference atp<0.001 against morbidity and mortality, and a significant difference atp<0.001 in the number of birds with CIA gross lesions scores. Resultsand statistical evaluations are given in Tables 10, 11, 12 and 13.Please note that one chick from the CIAV vaccinated group died 3 dpc andanother at 8 dpc. The chicks had no gross lesions or clinical signs ofCIAV. Therefore, mortality was ruled due to non-CIAV related causes.

Summary

These studies demonstrated that CIAV maternal antibody providedsignificant protection against CIA at p<0.05 to progeny of SPF whiteleghorn type chickens, which were previously vaccinated at 9 weeks ofage with the live chicken infectious anemia virus vaccine administratedvia the drinking water. The protection was assessed on the basis ofclinical signs, morbidity/mortality, and CIAV specific lesions atnecropsy. These studies demonstrated that maternal antibody protectionwas provided to chicks by hens through at least 37 weeks of age (28weeks post vaccination). TABLE 1 Pre-vaccination Serological Results byELISA of 9-week-old SPF Chickens to Confirm Negative Serological StatusPrior to Vaccination with Water-administered CIAV Vaccine. CIAVSerological Bird No. Band No. S/N Ratio by ELISA Status 1 104 0.89Negative^(a) 2 108 0.90 Negative 3 128 1.00 Negative 4 133 0.95 Negative5 141 0.98 Negative 6 190 0.84 Negative 7 191 0.95 Negative 8 201 0.89Negative 9 215 1.00 Negative 10 217 0.85 Negative 11 742 0.91 Negative12 747 0.89 Negative 13 753 0.82 Negative 14 765 0.91 Negative 15 7680.82 Negative 16 826 0.97 Negative 17 838 0.89 Negative 18 850 0.91Negative 19 856 0.86 Negative 20 866 0.98 Negative^(a)Negative = S/N Ratio >0.6 (IDEXX Kit interpretation)

TABLE 2 Clinical Gross Lesion Scores Hematocrit Values Signs Bone Bird14 21 Morbidity/ Mar- Thy- Mus- No. dpc^(a) dpc Mortality Liver row muscle 7 33 33 N/N 0 0 0 0 8 37 NS N/N 0 0 0 0 9 27 30 N/N 0 0 0 0 10 28 32N/N 0 0 0 0 11 34 27 N/N 0 0 0 0 12 31 40 N/N 0 0 0 0 13 34 26 N/N 0 0 00 14 26 26 N/N 0 0 0 0 15 28 31 N/N 0 0 0 0 16 32 33 N/N 0 0 0 0 17 3534 N/N 0 0 0 0 18 32 29 N/N 0 0 0 0 19 35 32 N/N 0 0 0 0 20 NS 34 N/N 00 0 0 21 35 33 N/N 0 0 0 0 22 28 23 N/N 0 0 0 0 23 33 38 N/N 0 0 0 0 24NS 27 N/N 0 0 0 0 25 31 NS N/N 0 0 0 0 No. 0/21 1/22 0/24/0/24 0/24 0/240/24 0/24 Positive^(a)Days post challenge^(b)No sample^(c)N = negative^(d)0 = normal, 1 = slight, 2 = moderate, 3 = severe gross lesionsassociated with CIA

TABLE 3 Study 1 Hematocrit Values, Clinical Signs, Mortality and CIAGross Lesion Scores of Chicks from 27-week-old Non-vaccinated BreederChickens Challenged at Day of Age with CIAV (Positive Controls).Clinical Signs Gross Lesion Scores Hematocrit Values Morbidity/ BoneBird No. 14 dpc^(a) 21 dpc Mortality Liver Marrow Thymus Muscle 1 16^(b)ND^(c) N^(d)/P^(e) 1^(f) 3 3 2 2 12 ND N/P 0 3 3 0 3 25 20 N/N 0 0 1 0 430 17 N/N 0 2 0 0 5 18  6 P/N 1 3 3 1 6 10 ND N/P 0 3 3 2 7 28 13 P/N 12 2 0 8 33 24 N/N 0 2 2 0 9 22 ND N/P 0 3 3 0 10 25 10 N/N 0 2 3 1 11 2732 N/N 0 0 0 0 12 30 13 N/N 0 2 3 0 13 NS^(g) 15 N/N 0 3 1 0 14 20 29N/N 0 0 0 0 15 17 ND N/P 0 3 3 0 16 16 30 N/N 0 0 0 0 17 25 11 P/N 0 3 32 18 23 25 N/N 0 1 1 0 19 33 15 N/N 0 2 2 0 20 15 ND N/P 0 2 2 0 21 2916 N/N 0 2 2 0 22 25 31 N/N 0 0 0 0 23 23 13 P/N 2 3 3 2 24 44 20 N/N 02 1 0 25 25 21 N/N 0 0 2 0 26 35 23 N/N 0 0 1 0 27 12 20 N/N 0 2 2 1 2830 24 N/N 0 0 0 0 29 33 18 N/N 0 2 2 0 30 25 39 N/N 0 0 0 0 31 25 ND P/P0 2 2 1 32 22 ND N/P 0 1 2 0 33 26 ND N/P 0 3 3 0 34 20 25 N/N 0 1 1 035 17 15 N/N 0 1 2 0 36 33 28 N/N 0 0 0 0 37 31 15 N/N 0 2 2 0 38 25 NDN/P 0 0 2 0 39 NS 27 N/N 0 0 1 0 40 30 24 N/N 0 1 0 0 No. 23/38 23/305/40/ 4/40 28/40 31/40 8/40 Positive 10/40 No. Birds with CIA No. Deador No. Birds with CIA No. Birds Positive for Positive Hematocrit Morbid= 14/40 Gross Lesion CIA/Total = 38/40 Values^(b)/Total = 36/40 (35.0%)Scores ≧1/ (95.0%) (90.0%) Total = 33/40 (82.5%)^(a)Days post challenge^(b)Hematocrit values ≦25 = anemia^(c)Not Done^(d)N = negative^(e)P = positive for clinical signs or CIAV mortality^(f)0 = normal, 1 = slight, 2 = moderate, 3 = severe gross lesionsassociated with CIA^(g)No sample

TABLE 4 Study 1 Hematocrit Values, Clinical Signs, Mortality and CIAGross Lesion Scores of Chicks with Maternal Antibody from 27-Week-oldCIAV Vaccinated Breeder Chickens Challenged at Day of Age with CIAV.Clinical Signs Gross Lesion Scores Hematocrit Values Morbidity/ BoneBird No. 14 dpc^(a) 21 dpc Mortality Liver Marrow Thymus Muscle 1 23^(b)32 N^(c)/N 0 0 0 0 5 ND^(d) ND N/Q^(e) 0 0 0 0 6 38 27 N/N 0 0 0 0 7 3328 N/N 0 0 0 0 9 29 30 N/N 0 0 0 0 10 29 30 N/N 0 0 0 0 11 30 30 N/N 0 00 0 12 29 29 N/N 0 0 0 0 13 27 39 N/N 0 0 0 0 14 34 34 N/N 0 0 0 0 19 2826 N/N 0 0 0 0 20 31 39 N/N 0 0 0 0 21 30 NS^(f) N/N 0 2^(g) 2 0 23 3434 N/N 0 0 0 0 24 35 28 N/N 0 0 0 0 25 30 26 N/N 0 0 0 0 28 30 27 N/N 00 0 0 29 29 28 N/N 0 0 0 0 30 35 35 N/N 0 0 0 0 33 32 33 N/N 0 0 0 0 3427 35 N/N 0 0 0 0 35 23 24 N/N 0 2 2 0 38 16 NS N/P^(h) 0 3 2 2 39 15 33N/N 0 0 0 0 40 27 35 N/N 0 0 0 0 No. 4/24 1/22 0/25 2/25 0/25 3/25 3/251/25 Positive No. Birds with CIA Positive No. Dead or No. Birds with CIANo. Birds CIA Hematocrit Values^(b)/Total = 4/24 Morbid 2/25 GrossLesion Positive/Totals 5/25 (16.7%) (8.0%) Scores ≧1/ (20.0%) Total =3/25 (12.0%)^(a)Days post challenge^(b)Hematocrit values ≦25 = anemia^(c)N = negative^(d)Not done^(e)Q = non CIAV associated mortality^(f)No serum^(g)0 = normal, 1 = slight, 2 = moderate, 3 = severe gross lesionsassociated with CIA^(h)P = positive for clinical signs or CIAV mortality

TABLE 5 Study 1 Summary of Hematocrit Values of CIAV Challenged Chicksfrom 27-Week-old Vaccinated and Non-vaccinated Breeder Chickens. No.Birds with No. Birds with No. Birds with Hematocrit ≦ 25 at Hematocrit ≦25 at Hematocrit ≦ 25 at 14 dpc/Total No. 21 dpc/Total No. either 14 or21 dpc/Total Group Evaluated Evaluated No. Evaluated Negative 0/21  1/22   1/24   Control (4.5%)   (4.2%)   Positive Control 23/38   23/30    36/40    (60.5%)    (76.7%)    (90.0%)    Progeny from 4/24  1/22   4/24   CIAV (16.7%)    (4.5%)   (16.7%)    Vaccinated HensFisher's Exact <0.001^(a)   <0.001^(a)   <0.001^(a)   Test p =^(a)Statistical difference by Fisher's Exact Test at p < 0.001 betweenpositive controls and progeny from CIAV vaccinated breeder chickens.

TABLE 6 Study 1 Summary of Clinical Signs and Mortality of CIAVChallenged Chicks from 27 Week-old Vaccinated and Non-vaccinated BreederChickens. No. with Clinical No. with Clinical No. Signs or GroupSigns/Total Dead/Total Dead/Total Negative 0/24  0/24   0/24   ControlPositive 5/40  10/40    14/40    Control (12.5%)   (25.0%)    (35.0%)   Progeny from 0/25  2/25   2/25   CIAV Vaccinated (8.0%)   (8.0%)   HensFisher's Exact 0.08  0.08    0.012^(a) Test p =^(a)Statistical difference by Fisher's Exact Test at p < 0.05 betweenpositive control group and progeny from CIAV vaccinated breederchickens.

TABLE 7 Study 1 Summary of CIA Gross Lesion Scores of CIAV ChallengedProgeny from 27-Week-old CIAV Vaccinated and Non-vaccinated BreederChickens. No. Birds with Gross Lesion Scores ≧1 (GLS)/ Total No. Birdsat Post-mortem No. Birds Bone with GLS ≧ Group Liver Marrow ThymusMuscle 1/Total Negative 0/24 0/24 0/24 0/24 0/24 Control Positive 4/4028/40  31/40  8/40 33/40  Control (10.0%) (70.0%) (77.5%) (20.0%)(82.5%) Progeny 0/25 3/25 3/25 1/25 3/25 from CIAV (12.0%) (12.0%)(4.0%) (12.0%) Vaccinated Hens Mann-Whitney 0.5 <0.001^(a) <0.001^(a)0.29 <0.001^(a) Test p = Fisher's Exact NA^(b) NA NA NA <0.001^(c) Testp =^(a)Statistical difference at p < 0.001 between positive control groupand progeny from CIAV vaccinated hens by Mann-Whitney Test.^(b)Not applicable.^(c)Statistical difference at p < 0.001 between positive control groupand progeny from CIAV vaccinated hens by Fisher's Exact Test.

TABLE 8 Study 2 Hematocrit Values, Clinical Signs, Mortality and CIAGross Lesion Scores of Non-challenged Chicks from 37-Week-oldNon-Vaccinated Breeder Chickens (Negative Controls). Clinical SignsHematocrit Values Morbidity/ Gross Lesion Scores Bird No. 14 dpc^(a) 21dpc Mortality Liver Bone Marrow Thymus Muscle 1 31 34 N/N^(b) 0^(c) 0 00 2 35 33 N/N 0 0 0 0 3 35 37 N/N 0 0 0 0 4 NS^(d) 35 N/N 0 0 0 0 5 3534 N/N 0 0 0 0 6 34 33 N/N 0 0 0 0 7 34 31 N/N 0 0 0 0 8 33 34 N/N 0 0 00 9 35 35 N/N 0 0 0 0 10 38 33 N/N 0 0 0 0 11 37 NS N/N 0 0 0 0 12 34 NSN/N 0 0 0 0 13 36 35 N/N 0 0 0 0 14 38 33 N/N 0 0 0 0 15 36 NS N/N 0 0 00 16 NS 35 N/N 0 0 0 0 17 35 NS N/N 0 0 0 0 18 33 35 N/N 0 0 0 0 19 4238 N/N 0 0 0 0 20 32 35 N/N 0 0 0 0 21 35 37 N/N 0 0 0 0 22 30 31 N/N 00 0 0 23 35 34 N/N 0 0 0 0 24 33 35 N/N 0 0 0 0 25 34 34 N/N 0 0 0 0 No0/23 0/21 0/25/0/25 0/25 0/25 0/25 0/25 positive^(a)Days post challenge^(b)N = negative^(c)0 = normal, 1 = slight, 2 = moderate, 3 = severe gross lesionsassociated with CIA^(d)No sample

TABLE 9 Study 2 Hematocrit Values, Clinical Signs, Mortality and CIAGross Lesion Scores of Chicks from 37-Week-old Non-Vaccinated BreederChickens Challenged at Day of Age with CIAV (Positive Controls).Clinical Signs Gross Lesion Scores Bird Hematocrit Values Morbidity/Bone No. 14 dpc^(a) 21 dpc Mortality Liver Marrow Thymus Muscle 1 30 20N/N^(b) 0 0 0 0 2 25^(c) 33 N/N 0 0 0 0 3 NS^(d) 30 N/N 0 0 0 0 4 29ND^(e) N/P^(f) 0 3^(g) 3 0 5 19 12 N/N 0 2 1 0 6 29 23 N/N 0 2 2 0 7 2715 N/N 0 3 2 0 8 14 13 N/N 0 0 3 0 9 18 ND N/P 0 3 3 0 10 25 20 N/N 0 10 0 11 33 22 N/N 0 0 0 0 12 21 ND P/P 0 3 3 0 13 13 23 P/N 0 2 0 0 14 2723 N/N 0 0 2 0 15 20 ND N/P 0 3 3 0 16 22 ND N/P 0 2 3 0 17 24 ND N/P 03 2 0 18 20 23 P/N 0 1 2 0 19 14 ND N/P 0 3 3 0 20 24 18 P/N 0 2 3 0 21 8 ND P/P 0 3 3 0 22 15 16 N/N 0 3 3 0 23 24 30 N/N 0 0 0 0 24 27 ND N/P0 3 3 0 25 27 29 N/N 0 0 0 0 26 14 15 N/N 0 2 3 0 27 23 ND N/P 0 3 3 028 13 32 N/N 0 0 0 0 29 25 31 N/N 0 0 0 0 30 22 28 N/N 0 0 0 0 31 NS NDN/P 0 2 3 0 32 35 30 N/N 0 0 0 0 33 24 22 P/N 0 1 2 0 34 17 26 N/N 0 0 00 35 29 29 N/N 0 0 0 0 36 25 18 N/N 0 0 0 0 37 27 35 N/N 0 0 0 0 38 2330 N/N 0 0 0 0 39 20 28 N/N 0 0 0 0 40 18 ND N/P 0 3 3 0 No. 27/38 15/286/40/12/40 0/40 22/40 22/40 0/40 Positive No. Birds with CIA PositiveNo. Dead or No. Birds with CIA No. Birds Positive Hematocrit Morbid =16/40 Gross Lesion for CIA/Total = Values^(c)/Total = 32/39 (82.1%)(40.0%) Scores ≧ 35/40 (87.5%) 1/Total = 24/40 (60.0%)^(a)Days post challenge^(b)N = negative^(c)Hematocrit values ≦25 = anemia^(d)No sample^(e)Not Done^(f)P = positive for clinical signs or CIAV mortality^(g)0 = normal, 1 = slight, 2 = moderate, 3 = severe gross lesionsassociated with CIA

TABLE 10 Study 2 Hematocrit Values, Clinical Signs, Mortality and CIAGross Lesion Scores of Chicks with Maternal Antibody from 37-Week-oldCIAV Vaccinated Breeder Chickens Challenged at Day of Age with CIAV.Clinical Signs Gross Lesion Scores Bird Hematocrit Values Morbidity/Bone No. 14 dpc^(a) 21 dpc Mortality Liver Marrow Thymus Muscle 1 30NS^(b) N/N^(c) 0 0 0 0 2 31 33 N/N 0 0 0 0 3 27 36 N/N 0 0 0 0 4 30 36N/N 0 0 0 0 5 33 ND^(d) N/P^(e) 0 2^(f) 3 0 6 30 40 N/N 0 0 0 0 7 27 40N/N 0 0 0 0 8 29 40 N/N 0 0 0 0 9 30 32 N/N 0 0 0 0 10 28 20^(g) N/N 0 00 0 11 27 34 N/N 0 0 0 0 12 38 39 N/N 0 0 0 0 13 NS 38 N/N 0 0 2 0 14 2041 N/N 0 0 0 0 15 21 43 N/N 0 0 0 0 16 35 40 N/N 0 0 0 0 17 31 32 N/N 00 0 0 18 29 39 N/N 0 0 0 0 19 35 46 N/N 0 0 0 0 20 23 38 N/N 0 0 0 0 2130 NS N/N 0 0 0 0 22 26 38 N/N 0 0 0 0 23 NS 40 N/N 0 0 0 0 24 33 43 N/N0 0 0 0 25 42 38 N/N 0 0 0 0 26 30 32 N/N 0 0 0 0 27 30 40 N/N 0 0 0 028 25 44 N/N 0 0 0 0 29 27 41 N/N 0 0 0 0 30 33 35 N/N 0 0 0 0 31 36 43N/N 0 0 0 0 32 30 41 N/N 0 0 0 0 33 32 38 N/N 0 0 0 0 34 ND ND N/Q^(h) 00 0 0 35 30 30 N/N 0 0 0 0 36 31 41 N/N 0 0 0 0 37 42 35 N/N 0 0 1 0 3834 36 N/N 0 0 0 0 39 27 35 N/N 0 0 0 0 40 ND ND N/Q^(h) 0 0 0 0 No. 4/361/35 0/40/3/40 0/40 1/40 3/40 0/40 positive No. Birds with CIA No. Deador Morbid No. Birds with CIA No. Birds CIA Positive Hematocrit 3/40(7.5%) Gross Lesion Positive/Total = Values^(g)/Total = 5/38 Scores ≧ 1/8/40(20.0%) (13.2%) Total = 3/40 (7.5%)^(a)Days post challenge^(b)No serum^(c)N = negative^(d)Not done^(e)P = positive for clinical signs and CIAV mortality^(f)0 = normal, 1 = slight, 2 = moderate, 3 = severe gross lesionsassociated with CIA^(g)Hematocrit values ≦ 25 = anemia^(h)Q = non CIAV associated mortality

TABLE 11 Study 2 Summary of Hematocrit Values of CIAV Challenged Chicksfrom 37-week-old Vaccinated and Non-vaccinated Breeder Chickens. No.Birds with No. Birds with No. Birds with Hematocrit ≦25 at Hematocrit≦25 a 14 Hematocrit ≦25 at 21 either 14 or 21 dpc/Total dpc/Total No.dpc/Total No. Group No. Evaluated Evaluated Evaluated Negative 0/23  0/21   0/25   Control Positive 27/38    15/28    32/39    Control(71.1%)    (53.6%)    (82.1%)    Progeny from CIAV 4/36   1/35   5/38  Vaccinated Hens (11.1%)    (2.9%)   (13.2%)    Fisher's Exact <0.001^(a)  <0.001^(a)   <0.001^(a)   Test p =^(a)Statistical difference by Fisher's Exact Test at p < 0.001 betweenpositive controls and progeny from CIAV vaccinated breeder chickens.

TABLE 12 Study 2 Summary of Clinical Signs and Mortality of CIAVChallenged Chicks from 37-week-old Vaccinated and Non-vaccinated BreederChickens. No. with Clinical No. with Clinical Group Signs/Total No.Dead/Total Signs or Dead/Total Negative Control 0/25 0/25 0/25 PositiveControl 6/40 (15%) 12/40 (30%) 16/40 (40.0%) Progeny from CIAVVaccinated Hens 0/40 3/40 (7.5%) 3/40 (7.5%) Fisher's Exact Test p =0.013^(a) 0.010^(a) <0.001^(a)^(a)Statistical difference by Fisher's Exact Test at p < 0.05 betweenpositive control group and progeny from CIAV vaccinated breederchickens.

TABLE 13 Study 2 Summary of CIA Gross Lesion Scores of CIAV ChallengedProgeny from 37-Week-old CIAV Vaccinated and Non-vaccinated BreederChickens. No. Birds with Gross Lesion Scores (GLS) ≧ 1/Total No. Birdsat Post-mortem No. Birds Bone with GLS ≧ Group Liver Marrow ThymusMuscle 1/Total Negative Control 0/25 0/25 0/25 0/25 0/25 PositiveControl 0/40 22/40 (55%) 22/40 (55%) 0/40 24/40 (60%) Progeny from 0/401/40 (2.5%) 3/40 (7.5%) 0/40 3/40 (7.5%) CIAV Vaccinated HensMann-Whitney 0.500 <0.001^(a) <0.001^(a) 0.293 <0.001^(a) Test p =Fisher's Exact NA^(b) NA NA NA <0.001^(c) Test p =^(a)Statistical difference at p < 0.001 between positive control groupand progeny from CIAV vaccinated hens by Mann-Whitney Test.^(b)Not applicable.^(c)Statistical difference at p < 0.001 between positive control groupand progeny from CIAV vaccinated hens by Fisher's Exact Test.

Example 8 Evaluation of Tumorgenicity in Chickens Following VariousTreatments on MDCC-MSB-1 Cells to Inactivate Marek's Virus Introduction

A tumorigenicity study was conducted on the MDCC-MSB-1 cell linesubstrate used for propagation of the Del-Ros strain of CIAV. Theobjective of this study was to demonstrate that a cell-free supernatantfluid derived from actively growing cell cultures lack the ability toinduce Marek's Disease (MD) tumors when inoculated into susceptiblechickens.

Materials and Methods

Groups of 25 to 36, SPF white leghorns chicks, aged 1-5 days wereinoculated with various inocula as shown in Table 1.

Chicks of both trials were observed daily for clinical signs of MD, andthe dead birds were necropsied and examined for gross lesions of MDduring a 8 week observation period. At the end of the observationperiod, all of the remaining birds (including the negative controls)were sacrificed with CO₂ and examined for MD related gross lesions.Samples of questionable or suspicious lesions were collected in 10%formaldehyde solution for histopathological examination.

Results

The MSB-1 cells without an additional processing step at a dose of 1×10⁶viable cells induced tumors in 2 of 36 chickens. However, additionallyprocessed cell free media did not induce tumors in chickens. The resultsare summarized in Table 2.

Summary

The data obtained from this study indicate that if MSB-1 cells are usedas the substrate for virus production such as for CIAV, it is necessaryto remove MSB-1 cells from the harvested virus to prevent the potentialof Marek's disease in chickens receiving the CIAV vaccine. Removal ofthe cells can be accomplished by filtering the MSB-1 virus infectedcells through a coarse filter (5 u size Millipore) to remove the cells.The cell-free virus fluid would be safe for to administer to chickens.

The results of this study demonstrated that additional processing stepsof the live virus (i.e., natural sedimentation followed by filtrationthrough 5 u Millipore filter) of the MSB-1 cells eliminates thepossibility of a vaccine produced in this cell line from inducing any MDrelated tumors in chickens.

The results suggest that filtration of the supernatant fluid of chickenanemia virus produced in MSB-1 cells will prevent the associated risk ofMD tumor formation when administered to chickens. TABLE 1 Experimentaldesign for the MSB-1 in-vivo tumorigenicity test: Dose of Group Route ofInoculum/ No. Treatments Total no. of chicks Inoculation Chick 1. ˜1.0 ×10⁶ viable MSB-1 cells 36 SQa 0.2 ml grown in RPMI 1640 mediumsupplemented with FBS. 2. Supernatant from a centrifuged 35 SQ 0.2 ml(2000 rpm for 10 min.) MSB-1 cell suspension. 3. RPMI 1640 medium 25 SQ0.2 ml supplemented with FBS (Medium control). 4. 3.0 × 10⁵ viable MSB-1cells/ml, 35 SQ 0.2 ml allowed to sediment naturally for overnight andthe resulting supernatant then filtered through 5u Millipore filter, andfinally treated at 41° C. for 24 hours before used for chickinoculation. 5. 3.0 × 10⁵ viable MSB-1 cells/ml, 35 SQ 0.2 ml allowed tosediment naturally for overnight and the resulting supernatant thenfiltered through 5 u Millipore filter before using for chickinoculation. 6. 3.0 × 10⁵ viable cells/ml, freeze 35 SQ 0.2 ml andthawed 3 times at −20° C. and then centrifuged at 2000 rpm for 15 min.,the resulting supernatant then filtered through 5 u Millipore filter,and lastly the filtrate was exposed to 41° C. for 24 hours before usingfor chick inoculation. 7.--- Negative controls 35 ND ND^(a)Subcutaneous

TABLE 2 Tumorigenicity test results of MDCC-MSB-1 cells. Necropsyresults (MD related Total Pos. % Test Total lesions) For MD MD groupsmortality Gross Histopath lesion Pos. Remarks 1 2/36 7 4 11 30.5 1 × 10⁶viable cells/chick indicates risk of MD tumor formation 2 0/35 2 3 514.3 Centrifuging at 2000 rpm for 15 min. is not enough to eliminatecells from the cell suspension, resulting in low incidence of tumorformation. 3 0/25 0 ND 0 0.0 The medium used for growing MSB-1 cells issafe for use 4 1/35 0 ND 0 0.0 Cell free filtrate does not induce tumor,safe for use in vaccine production 5 0/35 0 ND 0 0.0 Cell free filtratedoes not induce tumor, safe for use in vaccine production 6 0/35 0 ND 00.0 Cell free filtrate does not induce tumor, safe for use in vaccineproduction 7 0/35 0 ND 0 0.0 No tumors in the negative controls

Example 9 The Effects of Freeze-Thaw and 37° C. Incubation on theViability of Marek's Disease Virus Summary

Freeze-thaw up to 3 cycles could not completely inactivate Marek'sdisease virus (MDV) in tissue culture medium, but reduced the number ofplaques significantly. However, following 3 freeze-thaws and then 3days' incubation at 37° C, there was no MDV serotype 1 virus detected byIFA.

Introduction

Marek's disease virus and turkey herpesvirus (HVT) exist in eithercell-associated or cell free states, which have greatly differentsurvival properties. The infectivity of cell-associated virus stock isdirectly related to viability of the cells containing the virus. Theinfectivity of cell free virus preparation was reported to be sensitiveto different pH and temperatures. The viability of MDV, Rispen's strain,under freeze-thaw and 37° C. incubation treatments was investigated.

Materials and Methods

1. Cells: The CEF cells (primary CEF in roller bottle, secondary CEF in60 mm tissue culture plates) were prepared from 9 to 11 days-old SPFchicken embryos (SPAFAS).

2. Virus: The effect of freeze-thaw on the viability of Rispen's viruswas investigated by conducting an inactivation (kill) study. The activeRispen's infected CEF cells were harvested at 43 hpi. The infected cellswere resuspended in minimal essential medium (MEM) supplemented withfetal and calf sera and tryptose phosphate broth, and filled into 20tubes. The concentration of the cells was 36×10⁶ cells per ml. Sampleswere treated by freezing at −70° C. followed by thawing at roomtemperature, from one up to three cycles, then incubated at 37° C., fromone up to 15 days. The samples, with or without dilution, wereinoculated into secondary CEF monolayer in 60 mm tissue culture platesin duplicate, and incubated at 37° C. for 4-5 days. Titers were scoredby count plaques under a microscope with and without IFA stain with MDVserotype 1-specific monoclonal antibody 2BN90.

Results and Discussion

The MDV plaques were counted and reported as the average plaque formingunit (pfu) per ml. The results indicated that up to 3 freeze-thaw cyclesdid not completely inactivate MDV Rispen's strain in tissue culturemedium, but the number of plaques that indicated evidence of viablevirus were reduced significantly. However, with 3 or more daysincubation at 37° C. after 3 freeze-thaw cycles, there were no plaquesdetected by IFA (Table 1, and FIGS. 3 and 4), suggesting that combining3 freeze-thaw cycles with a 3-day incubation at 37° C. can completelydestroy MDV infectivity in the cell free medium. TABLE 1 The average MDVplaques resulting following each treatment Treatment Results Initialliter prior to freeze-thaw: 5.4 × 10⁶ pfu/ml Freeze-thaw once: 3 × 10⁴pfu/ml Freeze-thaw twice: 3 × 10³ pfu/ml (By IFA) Freeze-thaw 3 times:800 pfu/ml (By IFA) Freeze-thaw 3 times + 37° C. 1 day: 70 pfu/ml (ByIFA) Freeze-thaw 3 times + 37° C. 2 day: 25 pfu/ml (By IFA) Freeze-thaw3 times + 37° C. 3 day: 0 Freeze-thaw 3 times + 37° C. 4 day: 0Freeze-thaw 3 times + 37° C. 5 day: 0 Freeze-thaw 3 times + 37° C. 7day: 0 Freeze-thaw 3 times + 37° C. 9 day: 0 Freeze-thaw 3 times + 37°C. 11 day: 0 Freeze-thaw 3 times + 37° C. 13 day: 0 Freeze-thaw 3times + 37° C. 15 day: 0

Example 10 Comparison of Sequences for CIAV Strains

There are numerous reported strains of CIAV. Some of these have beensequenced and their sequences deposited. A chart comparing the aminoacid sequence of several of the known strains is provided below. It isbased on a pile up of sequences obtained from the NCBI database.Specific Amino Acid Changes in VP1, VP2 and VP3 of Several CAV IsolatesVPI Amino Acid Position Isolate Identification 14 84 92 144 157 229 251254 287 370 413 447 DRP5 5^(th) embryo passage Del-Ros strain A V G E VS R E S G S G DR Del-Ros strain A L G E V S R E S G S G TX Texas IsolateA L G E V F R E T G S T Cux-1 Cuxhaven-1 S L G D V S Q G A S A T IVIntervet Vaccine A L D E M S R G T S A T VP2 Amino Acid Position VP3Amino Acid Position Isolate Identification 153 169 4 23 73 103 116 118DRP5 5th embryo passage Del-Ros strain V D L R V S R C DR Del-Ros strainV D L R V S R C TX Texas Isolate V D L R V S R C Cux-1 Cuxhaven-1 A D LR V S K R IV Intervet Vaccine V G P Q A N R C

Nucleotide and amino acid sequences for the Del Ros strain are providedin the Sequence listing and also at NCBI accession no. AF313470.Nucleotide and amino acid sequences for additional other strains of CIAVcan be found as follows: intervet—NCBI accession no. D100068;Cuxhaven-1—NCBI accession no. NC001427; and CAV-15—NCBI accession no.AF372658. A nucleotide by nucleotide or amino acid by amino acidcomparison of these and other sequence can be routinely made.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1. A chicken infectious anemia virus (CIAV) vaccine, comprising liveCIAV passaged in MDCC-MSB-1 (MSB-1) cells, wherein the vaccine does notcause Marek's Disease.
 2. The CIAV vaccine of claim 1, wherein thevaccine does not produce gross lesions in chicken embryos.
 3. The CIAVvaccine of claim 1, wherein the vaccine does not produce anemia inchicken embryos.
 4. A method of making a CIAV vaccine, comprisingculturing CIAV in MSB-1 cells, and removing or killing any Marek'sdisease virus present in the CIAV-containing MSB-1 cell culture.
 5. Themethod of claim 4, comprising subjecting the CIAV-containing MSB-1 cellculture to at least 3 cycles of freezing and thawing, followed by a stepof maintaining the cells for about 3 days at about 37° C.
 6. The methodof claim 4, comprising the step of filtering the MSB-1 cell culturethrough a 5 micron filter.
 7. The method of claims 5 or 6, wherein themethod makes a vaccine that does not cause Marek's disease in chickensimmunized with the vaccine.
 8. A method of immunizing a chicken againstCIAV infection, comprising administering to the chicken an amount of theCIAV vaccine of claim 1 sufficient to induce an immune response to CIAV.9. The method of claim 8, wherein the immune response is protectiveagainst infection by CIAV.
 10. The method of claim 8, wherein the immuneresponse is protective against clinical disease caused by CIAVinfection.
 11. The method of claim 8, wherein the immune responseproduces antibodies that are protective against CIAV infection in theprogeny of immunized chickens.
 12. The method of claim 8, wherein thevaccine is administered to chickens from about 4 to 12 weeks of age. 13.The method of claim 8, wherein the vaccine is administered in drinkingwater.
 14. The method of claim 8, wherein the vaccine is administered byparenterally.
 15. The method of claim 14, wherein the vaccine isadministered by spray.
 16. The method of claim 14, wherein the vaccineis administered by injection.